The long-term objective of this proposal is to provide a fundamental understanding of the cell biology of intracellular protein trafficking in polarized epithelial cells, focusing particularly on the role of the heterotrimeric G protein, Gi3. We have shown that the alpha subunit of Gi3, G alpha i3, can regulate, through as yet poorly defined biochemical pathways, membrane protein trafficking at the level of the Golgi in the basolateral secretory pathway. We propose that this action requires a series of protein interactions mediated by complex structural motifs in G alpha i3 and auxiliary proteins. The initial aims of this proposal are to define the structural motifs of G alpha i3 based on the recently solved crystal structure of other G alpha proteins, that are important for 1) proper targeting to Golgi membranes; and 2) binding to GAIP, a recently identified G alpha i3 binding protein of the RGS family. We propose to generate mutants of G alpha i3 (especially in the Insert 3 Loop) and GAIP and to study their intracellular targeting utilizing immunofluorescence, confocal, and electron microscopy and to study their ability to bind to each other using glutathione-S- transferase fusion protein column. We have shown that GAIP prefers the GTP-bound form of G alpha i3; we will not determine whether the interaction of GAIP with G alpha i3-GTP results in enhanced GTP hydrolysis. The effects of G alpha i3 and GAIP will determine in 1) secretion assays in non-polar and epithelial cells, and in semi-intact cells using endogenous and reported proteins as monitors of secretion; and in 2) protein trafficking assays utilizing fluorescence recovery after photobleaching for mennosidase II/green fluorescent protein (GFP) as a marking for intra-Golgi protein cycling, TGN38-GFP as a marker for trans-Golgi network and basolateral membrane cycling, and growth hormone-GFP to monitor the secretory pathway. In addition, the role of G alpha i3 and GAIP in endocytosis will be quantitated utilizing a fluorescence assay of intracellular vesicle fusion. Finally, the signal transduction pathway for G alpha i3 at the level of the Golgi will be explored by monitoring the response of the MAP kinase cascade as a function of G alpha i3 action and by purifying and cloning G alpha i3 effector proteins. The secretory pathway is of fundamental importance to cell growth, development, and function, and in epithelia to the development of polarity. Understanding the molecular mechanisms for its regulation will provide new opportunities for modulating cellular responses in health and disease.